Earlier studies have shown that the comonomer triethyleneglycol-dimethacrylate (TEGDMA) and the photostabilizer 2-hydroxy-4-methoxybenzophenone (HMBP) are cytotoxic and inhibit cell growth. It was the aim of this study to elucidate the underlying metabolic effects of TEGDMA and HMBP on immortal contact-inhibited Swiss albino mouse embryo cells (3T3 fibroblasts) by nuclear magnetic resonance (NMR) spectroscopy. Cell extracts and culture media were analyzed by NMR spectroscopy for metabolic changes after incubation for 24 hours with ED20-concentrations of TEGDMA and HMBP. TEGDMA could be detected in all fractions (cytosol, lipid fractions, and culture media) of 3T3 cells, while HMBP was found only in the lipid fraction accumulated at a maximum rate (51 nmol/mg DNA) compared with TEGDMA (27 nmol/mg DNA). TEGDMA increased the concentration of phosphomonoesters to 180 ± 36% and decreased the phosphodiesters to 65 ± 5% of controls (control = 100%). Thus, the turnover of phospholipids was enhanced, whereas content and composition of phospholipids of membranes did not alter markedly. Additionally, TEGDMA changed the metabolic state of cells, indicated by slight decreases of nucleoside triphosphates and an increase in the ratio of nucleoside diphosphates to nucleoside triphosphates, while HMBP had no effect. The most remarkable effect of TEGDMA was a nearly complete decline of the intracellular glutathione levels. Analysis of our data shows that NMR spectroscopy of cell-material interactions may reveal metabolic effects of organic test substances which are not detectable by standard in vitro assays. The comonomer TEGDMA affected the metabolism of the cells on different levels, while HMBP accumulated in the lipid fraction and induced significantly fewer effects on cell metabolism.
Bean TA, Zhuang WC, Tong PY, Eick JD, Yourtee DM (1994). Effect of esterase on methacrylates and methacrylate polymers in enzyme simulator for biodurability and biocompatibility testing . J Biomater Res28:59-63.
2.
Blaheta RA, Franz M., Auth Mkh, Markus BH (1993). Determination of cell counts and cell proliferation by the fluorochromes Hoechst 33342 or Hoechst 33258. In: Cell and tissue culture models in dermatological research. Bernd A, Bereiter-Hahn J, Hevert F, Holzmann H, editors. Berlin: Springer-Verlag.
3.
Bligh EG, Dyer WJ ( 1959). A rapid method of total lipid extraction and purification . Can J Biochem Physiol37:911-917.
4.
Burton K. (1955). A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J62:315-323.
5.
Caughman GB, Schuster GS, Rueggeberg FA (1999). Cell lipid alterations resulting from prolonged exposure to dimethylaminoethylmethacrylate. Clin Oral Invest3:181-187.
6.
Cohen JS, Lyon RC, Daly PF ( 1989). Monitoring intracellular metabolism by nuclear magnetic resonance. Meth Enzymol177:435-452.
7.
Daly PF, Cohen JS (1989). Magnetic resonance spectroscopy of tumors and potential in vivo clinical applications: a review. Cancer Res49:770-779.
8.
de Certaines JD (1996). High resolution nuclear magnetic resonance spectroscopy in clinical biology: application in oncology. Anticancer Res16:1325-1331.
9.
Deneke SM, Fanburg BL (1989). Regulation of intracellular glutathione. Am J Physiol257:163-173.
10.
Edzes HT, Teerlink T., Valk J. ( 1991). Phospholipid identification in tissue extracts by two-dimensional 31P-1H NMR spectroscopy with isotropic proton mixing. J Magnet Res95:594-602.
11.
el Dareer SM, Kalin JR, Tillery KF, Hill DL ( 1986). Disposition of 2-hydroxy-4-methoxybenzophenone in rats dosed orally, intravenously, or topically. J Toxicol Environ Health19:491-502.
12.
Elovaara E., Kivisto H., Vainio H. (1983). Effects of methylmethacrylate on non-protein thiols and drug metabolizing enzymes in rat liver and kidneys. Arch Toxicol52:109-121.
13.
Engelmann J., Henke J., Willker W., Kutscher B., Nossner G., Engel J., et al. (1996). Early stage monitoring of miltefosine induced apoptosis in KB cells by multinuclear NMR spectroscopy. Anticancer Res16:1429-1440.
14.
Flogel U., Niendorf T., Serkova N., Brand A., Henke J., Leibfritz D. (1995). Changes in organic solutes, volume, energy state, and metabolism associated with osmotic stress in a glial cell line: a multinuclear NMR study. Neurochem Res20:793-802.
15.
Fujisawa S., Kadoma Y., Masuhara E. (1984). A calorimetric study of the interaction of synthetic phospholipid liposomes with vinyl monomers, acrylates and methacrylates. J Biomed Mater Res18:1105-1114.
16.
Fujisawa S., Kadoma Y., Kadoma Y. (1988). 'H and 13C NMR studies of the interaction of eugenol, phenol, and triethyleneglycol dimethacrylate with phospholipid liposomes as a model system for odontoblast membranes. J Dent Res67:1438-1441.
17.
Fujisawa S., Atsumi T., Kadoma Y. (2000). Cytotoxicity of methyl methacrylate (MMA) and related compounds and their interaction with dipalmitoylphosphatidylcholine (DPPC) liposomes as a model for biomembranes. Oral Dis6:215-221.
18.
Geurtsen W. (1998). Substances released from dental resin composites and glass ionomer cements. Eur J Oral Sci106:687-695.
19.
Geurtsen W. (2000). Biocompatibility of resin-modified filling materials . Crit Rev Oral Biol Med11:333-355.
20.
Geurtsen W., Lehmann F., Spahl W., Leyhausen G. (1998a). Cytotoxicity of 35 dental resin composite monomers/additives in permanent 3T3 and three human primary fibroblast cultures. J Biomed Mater Res41:474-480.
21.
Geurtsen. W., Spahl W., Leyhausen G. (1998b). Residual monomer/additive release and variability in cytotoxicity of light-curing glass-ionomer cements and compomers. J Dent Res77:2012-2019.
22.
Geurtsen W., Spahl W., Leyhausen G. (1999a). Variability of cytotoxicity and leaching of substances from four light-curing pit and fissure sealants. J Biomed Mater Res44:73-77.
23.
Geurtsen W., Spahl W., Müller K., Leyhausen G. (1999b). Aqueous extracts from dentin adhesives contain cytotoxic chemicals. J Biomed Mater Res48:772-777.
24.
Goa J. (1953). A micro biuret method for protein determination . Scan J Clin Lab Invest5:218-222.
25.
Hanks CT, Craig RG, Diehl ML, Pashley DH (1988). Cytotoxicity of dental composites and other materials in a new in vitro device. J Oral Pathol17:396-403.
26.
Hanks CT, Strawn SE, Wataha JC, Craig RG (1991). Cytotoxic effects of resin components on cultured mammalian fibroblasts. J Dent Res70:1450-1455.
27.
Hansel C., Leyhausen G., Mai Ueh, Geurtsen W. (1998). Effects of various resin composite (co)monomers and extracts on two caries-associated micro-organisms in vitro. J Dent Res77:60-67.
28.
Heil J., Reifferscheid G., Waldmann P., Leyhausen G., Geurtsen W. (1996). Genotoxicity of dental materials. Mutat Res368:181-194.
29.
Henke J., Willker W., Engelmann J., Leibfritz D. (1996). Combined extraction techniques of tumour cells and lipid/phospholipid assignment by two dimensional NMR spectroscopy. Anticancer Res16:1417-1427.
30.
Henke J., Engelmann J., Flogel U., Pfeuffer J., Kutscher B., Nossner G., et al. (1998). Apoptotic effects of hexadecylphosphocholine on resistant and nonresistant cells monitored by NMR spectroscopy. Drugs of Today34(Suppl F):37-50.
31.
Kaplan 0, van Zijl Pcm, Cohen JS (1992). NMR studies of metabolism of cells and perfused organs. NMR Basic Principles Progr28:3-52.
32.
Lefebvre CA, Schuster GS, Rueggeberg FA, Tamareselvy K., Knoernschild KL (1996). Responses of oral epithelial cells to dental resin components. J Biomater Sci Polym Edn7:965-976.
33.
Lehmann F., Leyhausen G., Spahl W., Geurtsen W. (1993). Comparative cell culture studies of the cytotoxicity of composite resin components. Dtsch Zahnärztl Z48:651-653 (in German).
34.
Leyhausen G., Heil J., Reifferscheid G., Geurtsen W. (1995). The genotoxic potential of composite components . Dtsch Zahnärztl Z50:134-136 (in German).
35.
Leyhausen G., Abtahi M., Karbakhsch M., Sapotnik A., Geurtsen W. (1998). The biocompatibility of various resin-modified and one conventional glass-ionomer cement. Biomaterials19:559-564.
36.
London RE (1988). 13C labeling in studies of metabolic regulation . Progr Nuclear Magnet Reson Spectrosc20:337-383.
37.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951). Protein measurement with the folin phenol reagent . J Biol Chem193:265-275.
38.
Lyon RC, Cohen JS, Faustino PJ, Megnin P., Myers CE (1988). Glucose metabolism in drug-sensitive and drug-resistant human breast cancer cells monitored by magnetic resonance spectroscopy. Cancer Res48:870-877.
39.
McCarthy TJ, Witz G. ( 1997). Structure-activity relationships in the hydrolysis of acrylate and methacrylate esters by carboxylesterase in vitro. Toxicology116:153-158.
40.
McCarthy TJ, Hayes EP, Schwartz CS, Witz G. ( 1994). The reactivity of selected acrylate esters toward glutathione and deoxyribonucleosides in vitro: structure-activity relationships. Fund Appl Toxicol22:543-548.
41.
Meister A. (1994). Glutathione, ascorbate, and cellular protection . Cancer Res54(Suppl):1969-1975.
42.
Meister A. (1995). Mitochondrial changes associated with glutathione deficiency. Biochim Biophys Acta1271:35-42.
43.
Meister A., Anderson ME (1983). Glutathione. Annu Rev Biochem52:711-760.
44.
Merchant TE, Gierke LW, Meneses P., Glonek T. (1988). 31P magnetic resonance spectroscopic profiles of neoplastic human breast tissues. Cancer Res48:5112-5118.
45.
Nassiri MR, Hanks CT, Cameron MJ, Strawn SE, Craig RG (1994). Application of flow cytometry to determine the cytotoxicity of urethane dimethacrylate in human cells. J Biomed Mater Res28:153-158.
46.
Negendank W. (1992). Studies of human tumors by MRS. NMR Biomed5:303-324.
47.
Okereke CS, Kadry AM, Abdel-Rahman MS, Davis RA, Friedman MA (1993). Metabolism of benzophenone-3 in rats. Drug Metab Dispos21:788-791.
48.
Okereke CS, Abdel-Rahman MS, Friedman MA (1994). Disposition of benzophenone-3 after dermal administration in male rats. Toxicol Lett73:113-122.
49.
Patel SS, Walt DR ( 1988). Acetyl coenzyme A synthetase catalyzed reactions of coenzyme A with alpha, beta-unsaturated carboxylic acids. Anal Biochem170:355-360.
Rathbun MA, Craig RG, Hanks CT, Filisko FE (1991). Cytotoxicity of a Bis-GMA dental composite before and after leaching in organic solvents. J Biomed Mater Res25:443-457.
52.
Rudkin TM, Arnold DL (1999). Proton magnetic resonance spectroscopy for the diagnosis and management of cerebral disorders. Arch Neurol56:919-926.
53.
Ruiz-Cabello J., Cohen JS (1992). Phospholipid metabolites as indicators of cancer cell function. NMR Biomed5:226-233.
54.
Schuster GS, Lefebvre CA, Dirksen DR, Knoemschild KL, Caughman GB (1995). Relationship between denture base resin in cytotoxicity and cell lipid metabolism. Int J Prosthodont8:580-586.
55.
Schuster GS, Erbland JF, Lefebvre CA, Caughman CB, Knoernschild KL (1997). Effects of an aminomethacrylate on the epithelial cell metabolism. J Biomater Sci Polym Educ8:363-375.
56.
Schweikl H., Schmalz G. (1999). Triethylene glycol dimethacrylate induces large deletions in the hprt gene of V79 cells. Mutat Res438:71-78.
57.
Schweikl H., Schmalz G., Rackebrandt K. (1998). The mutagenic activity of unpolymerized resin monomers in Salmonella typhimurium and V79 cells. Mutat Res415:119-130.
58.
Shaka AJ, Keeler J., Frenkiel T., Freeman R. (1983). An improved sequence for broadband decoupling: WALTZ 16. J Magnet Res52:335-338.
59.
Shimizu M., Hochadel JF, Waalkes MP (1997). Effects of glutathione depletion on cadmium-induced metallothionein synthesis, cytotoxicity, and proto-oncogene expression in cultured rat myoblasts. J Toxicol Environ Health51:609-621.
60.
Spahl W., Budzikiewicz H. (1994). Qualitative analysis by gas and liquid chromatography/mass spectrometry of dental resin composites. Fresenius J Anal Chem350:684-691.
61.
Spahl W., Budzikiewicz H., Geurtsen W. (1998). Determination of leachable components from four commercial dental composites by gas and liquid chromatography/mass spectrometry . J Dent26:137-145.
62.
Szwergold BS (1992). NMR spectroscopy of cells. Annu Rev Physiol54:775-798.
63.
Terakado M., Yamazaki M., Tsujimoto Y., Kawashima T., Nagashima K., Ogawa J., et al. (1984). Lipid peroxidation as a possible cause of benzoylperoxide toxicity in rabbit dental pulp-a microsomal lipid peroxidation in vitro. J Dent Res63:901-905.
64.
Theilig C., Tegtmeier Y., Leyhausen G., Geurtsen W. (2000). Effects of BisGMA and TEGDMA on proliferation, migration, and tenascin expression of human fibroblasts and keratinocytes . J Biomed Mater Res53:632-639.
65.
Tyagi RK, Azrad A., Degani H., Salomon Y. (1996). Simultaneous extraction of cellular lipids and water-soluble metabolites: evaluation by NMR spectroscopy. Magnet Reson Med35:194-200.
66.
Vankerckhoven H., Lamprechts P., van Beylen M., Vanherle G. (1981). Characterization of composite resins by NMR and TEM. J Dent Res60:1957-1965.
67.
Wataha JC, Hanks CT, Strawn SE, Fat JC (1994). Cytotoxicity of components of resins and other dental restorative materials. J Oral Rehabil21:453-462.
68.
Willker W., Engelmann J., Brand A., Leibfritz D. (1996). Metabolite identification in cell extracts and culture media by proton-detected 2D-H, C-NMR spectroscopy. J Magnet Reson Anal2:21-32.
69.
Zwingmann C., Brand A., Richter-Landsberg C., Leibfritz D. (1998). Multinuclear NMR studies on NH4Cl-induced metabolic alterations and detoxification processes in primary astrocytes and glioma cells. Dev Neurosci20:417-426.
